Chromosome translocations and aberrations are common in cancer, but the molecular mechanisms responsible for chromosome fragility are poorly understood. We are using the human RNU2 locus (encoding U2 snRNA) as a model system to understand the causes of metaphase chromosome fragility. The genes encoding U2 snRNA, a key component of the mRNA splicing apparatus, are organized as a single essentially perfect tandem array containing 5 to >22 copies of a 6.1 kb repeat unit on human chromosome 17q21. The regularity of the U2 tandem repeat, the wealth of functional data regarding U2 snRNA transcription, and the variety of conditions that induce RNU2 fragility, all make RNU2 a good system for studying the relationship between gene function and metaphase chromatin condensation. Most importantly, the U2 genes are the only experimental system in which a new fragile site can be created de novo by inserting a cloned fragile site (i.e. tandemly repeated U2 genes or mutants thereof) into a new chromosomal locus. We have shown previously that: active U2 snRNA transcription is required for RNU2 metaphase fragility; all conditions that induce fragility also induce p53; loss of CSB (Cockayne Syndrome Group B protein), a putative transcription elongation factor required for transcription-coupled repair (TCR), causes constitutive RNU2 fragility; and the C-terminal domain (CTD) of p53, which binds CSB, can itself induce fragility. The data suggest that activated p53 induces fragility of the U2 genes by modulating CSB function, and that CSB is required either for U2 transcription, or for repair of damaged U2 genes. We propose a multifaceted experimental approach to test, modify, or discard this working model. Specifically, we will examine the chromatin structure of actively transcribing U2 snRNA genes in Xenopus oocytes (Aim 1) and in human somatic cells (Aim 2); we will assay the function of CSB in U2 transcription (Aim 3) and in DNA repair (Aim 4) along with XPG, BRCA1, and BRCA2; we will assay modulation of CSB function by p53 (Aim 5); and we will ask by microarray technology what cellular mRNAs are affected by loss of CSB activity (Aim 6). Our studies could shed light on cell cycle control of transcription, higher order chromosome structure, the molecular mechanisms of chromosome instability in cancer, and the causes of Cockayne Syndrome.